A feline homeobox-containing DNA sequence was isolated from the genomic library of the domestic cat using the mouse homeobox Hox-3.1 probe. The isolated homeobox was named cat HOX3A, according to the human homeobox nomenclature. The nucleotide sequence and the deduced amino acid sequence of cat HOX3A showed a higher similarity to those of cognate genes of human, mouse, and rat. Using a panel of rodent x domestic cat somatic cell hybrids, cat HOX3A was assigned to cat chromosome B4. Interestingly, it has been known that cat chromosome B4 shows syntenic homology with mouse chromosome 15 and human chromosome 12, where mouse Hox-3.1 and human HOX3A have been mapped, respectively. These data demonstrate evolutionary conservation of both HOX3A gene sequences and chromosomal locations during mammalian evolution.